Bottom Line:
Spinal bone mineral density (BMD) and micro-architecture by microCT measurement progressively decreased and deteriorated from 8 to 10, 12 and 14 months old, which was more apparent in females than the male counterparts.The estrogenic effect of EE2 in O. latipes was confirmed by significant up-regulation of four key estrogen responsive genes in the liver.In general, bone histomorphometric analyses indicated significantly lowered osteoblasts and osteoclasts numbers and surfaces on vertebrae of EE2-fed medaka.

Affiliation: State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong.

ABSTRACT

Background: In human, a reduction in estrogen has been proposed as one of the key contributing factors for postmenopausal osteoporosis. Rodents are conventional models for studying postmenopausal osteoporosis, but the major limitation is that ovariectomy is needed to mimic the estrogen decline after menopause. Interestingly, in medaka fish (Oryzias latipes), we observed a natural drop in plasma estrogen profile in females during aging and abnormal spinal curvature was apparent in old fish, which are similar to postmenopausal women. It is hypothesized that estrogen associated disorders in bone metabolism might be predicted and prevented by estrogen supplement in aging O. latipes, which could be corresponding to postmenopausal osteoporosis in women.

Principal findings: In O. latipes, plasma estrogen was peaked at 8 months old and significantly declined after 10, 11 and 22 months in females. Spinal bone mineral density (BMD) and micro-architecture by microCT measurement progressively decreased and deteriorated from 8 to 10, 12 and 14 months old, which was more apparent in females than the male counterparts. After 10 months old, O. latipes were supplemented with 17α-ethinylestradiol (EE2, a potent estrogen mimic) at 6 and 60 ng/mg fish weight/day for 4 weeks, both reduction in spinal BMD and deterioration in bone micro-architecture were significantly prevented. The estrogenic effect of EE2 in O. latipes was confirmed by significant up-regulation of four key estrogen responsive genes in the liver. In general, bone histomorphometric analyses indicated significantly lowered osteoblasts and osteoclasts numbers and surfaces on vertebrae of EE2-fed medaka.

Significance: We demonstrate osteoporosis development associated with natural drop in estrogen level during aging in female medaka, which could be attenuated by estrogen treatment. This small size fish is a unique alternative non-mammalian vertebrate model for studying estrogen-related molecular regulation in postmenopausal skeletal disorders in vivo without ovariectomy.

pone-0088165-g004: Representative appearance of female (top) and male (bottom) Japanese medaka Oryzias latipes at 8, 10, 12 and 14 months old (Fig. A).(B) Representative micro-architecture of vertebrae bodies at 8, 10, 12 and 14 months in females (left) and males (right) by 3-dimensional microCT reconstruction. (C) Age-related changes in bone mineral density (BMD) from 8 to 14 months in females (left) and males (right) by microCT analysis. White arrows and arrowheads indicate micro-cracks and thinner arches found in 10–14 months old fish. BMD values labeled by the same letter (a, b, c, d) on the graph are not significantly different from each other (p<0.05).

Mentions:
The BMD and micro-architecture of 15th∼25th vertebrae bodies were measured by microCT for both genders at four different age groups (8, 10, 12 and 14 months old) (Fig. 4A). As revealed by three dimensional reconstruction images using microCT, the micro-architecture of the vertebrae bodies deteriorated, including the formation of micro-cracks (white arrows), thinner arches (hemal and neural) (white arrowheads) and progressively curved vertebral column (Fig. 4B), which was obvious in females at 12 months old onwards and in males at 14 months. The BMD in females declined from 8 to 10 months old and reached significantly lower level at 12 and 14 months (P<0.05) (Fig. 4C, left). In males, the BMD declined gradually with age and towards significantly lower level at 12 months onwards compared to 8 and 10 months (Fig. 4C, right). Age-associated decline of BMD was faster in females than males after 10 months old (Fig. 4C).

pone-0088165-g004: Representative appearance of female (top) and male (bottom) Japanese medaka Oryzias latipes at 8, 10, 12 and 14 months old (Fig. A).(B) Representative micro-architecture of vertebrae bodies at 8, 10, 12 and 14 months in females (left) and males (right) by 3-dimensional microCT reconstruction. (C) Age-related changes in bone mineral density (BMD) from 8 to 14 months in females (left) and males (right) by microCT analysis. White arrows and arrowheads indicate micro-cracks and thinner arches found in 10–14 months old fish. BMD values labeled by the same letter (a, b, c, d) on the graph are not significantly different from each other (p<0.05).

Mentions:
The BMD and micro-architecture of 15th∼25th vertebrae bodies were measured by microCT for both genders at four different age groups (8, 10, 12 and 14 months old) (Fig. 4A). As revealed by three dimensional reconstruction images using microCT, the micro-architecture of the vertebrae bodies deteriorated, including the formation of micro-cracks (white arrows), thinner arches (hemal and neural) (white arrowheads) and progressively curved vertebral column (Fig. 4B), which was obvious in females at 12 months old onwards and in males at 14 months. The BMD in females declined from 8 to 10 months old and reached significantly lower level at 12 and 14 months (P<0.05) (Fig. 4C, left). In males, the BMD declined gradually with age and towards significantly lower level at 12 months onwards compared to 8 and 10 months (Fig. 4C, right). Age-associated decline of BMD was faster in females than males after 10 months old (Fig. 4C).

Bottom Line:
Spinal bone mineral density (BMD) and micro-architecture by microCT measurement progressively decreased and deteriorated from 8 to 10, 12 and 14 months old, which was more apparent in females than the male counterparts.The estrogenic effect of EE2 in O. latipes was confirmed by significant up-regulation of four key estrogen responsive genes in the liver.In general, bone histomorphometric analyses indicated significantly lowered osteoblasts and osteoclasts numbers and surfaces on vertebrae of EE2-fed medaka.

Affiliation:
State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong.

ABSTRACT

Background: In human, a reduction in estrogen has been proposed as one of the key contributing factors for postmenopausal osteoporosis. Rodents are conventional models for studying postmenopausal osteoporosis, but the major limitation is that ovariectomy is needed to mimic the estrogen decline after menopause. Interestingly, in medaka fish (Oryzias latipes), we observed a natural drop in plasma estrogen profile in females during aging and abnormal spinal curvature was apparent in old fish, which are similar to postmenopausal women. It is hypothesized that estrogen associated disorders in bone metabolism might be predicted and prevented by estrogen supplement in aging O. latipes, which could be corresponding to postmenopausal osteoporosis in women.

Principal findings: In O. latipes, plasma estrogen was peaked at 8 months old and significantly declined after 10, 11 and 22 months in females. Spinal bone mineral density (BMD) and micro-architecture by microCT measurement progressively decreased and deteriorated from 8 to 10, 12 and 14 months old, which was more apparent in females than the male counterparts. After 10 months old, O. latipes were supplemented with 17α-ethinylestradiol (EE2, a potent estrogen mimic) at 6 and 60 ng/mg fish weight/day for 4 weeks, both reduction in spinal BMD and deterioration in bone micro-architecture were significantly prevented. The estrogenic effect of EE2 in O. latipes was confirmed by significant up-regulation of four key estrogen responsive genes in the liver. In general, bone histomorphometric analyses indicated significantly lowered osteoblasts and osteoclasts numbers and surfaces on vertebrae of EE2-fed medaka.

Significance: We demonstrate osteoporosis development associated with natural drop in estrogen level during aging in female medaka, which could be attenuated by estrogen treatment. This small size fish is a unique alternative non-mammalian vertebrate model for studying estrogen-related molecular regulation in postmenopausal skeletal disorders in vivo without ovariectomy.